Inhibition of activated protein C by recombinant alpha 1-antitrypsin variants with substitution of arginine or leucine for methionine358.
alpha 1-Antitrypsin (alpha 1-AT) was recently identified as a major physiologic plasma inhibitor of activated protein C. The reaction with activated protein C of recombinant alpha 1-AT containing amino acid substitutions at the reactive center was studied. The substitution of Arg358 for Met, as observed in a patient with a severe bleeding disorder with the mutant alpha 1-AT Pittsburgh, increased the association rate constant for activated protein C from 1.1 x 10(1) to 4.9 x 10(4) M-1 s-1. The association rate constant of activated protein C with protein C inhibitor, a native plasma serpin that contains Arg354 at the reactive site, is 6 x 10(3) M-1 s-1 in the absence of heparin. Plasma containing 4 microM [Arg358]alpha 1-AT inhibited activated protein C activity by greater than 95% in 15 s, and the inhibited activated protein C was shown by immunoblotting to exist as activated protein C-inhibitor complexes. In controls 50% loss of activated protein C activity in normal plasma occurred in 19 min. Double-substituted [Pro357,Met358]alpha 1-AT----[Ala357,Arg358]alpha 1-AT had similar reactivity toward activated protein C as the single-substituted [Arg358]alpha 1-AT. Thus, replacement of the reactive center Met358 of alpha 1-AT by Arg358, analogous to Arg354 of protein C inhibitor, results in an activated protein C inhibitor that is more potent than either of the native inhibitors. Comparison of the association rate constant of the [Arg358]alpha 1-AT for activated protein C to that for thrombin (4 x 10(4) versus 3 x 10(5) M-1 s-1) suggests that thrombin would be more effectively inhibited than activated protein C, thereby giving an explanation for bleeding rather than thrombosis in the alpha 1-AT Pittsburgh patient.
Heeb
M J
MJ
Committee on Vascular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037.
Bischoff
R
R
Courtney
M
M
Griffin
J H
JH
D001120:Arginine; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D014644:Genetic Variation; D006801:Humans; D007700:Kinetics; D007930:Leucine; D008715:Methionine; D011486:Protein C / Q000037:antagonists & inhibitors*; D011994:Recombinant Proteins / Q000494:pharmacology; D000515:alpha 1-Antitrypsin / Q000235:genetics / Q000494:pharmacology*
D011486:Protein C; D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin; D001120:Arginine; D008715:Methionine; D007930:Leucine